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J A DiPaolo

Publications and source records attributed to J A DiPaolo.

At least 19 recordsLinked to original sources
PubMed1996

Tissue-specific expression, evolutionary conservation and localization of the cph proto-oncogene on Syrian hamster chromosome X.

Treatment of Syrian hamster embryo fibroblasts with a single dose of 3-methylcholanthrene caused the activation of the transforming potential of cellular sequences (Notario et al, Oncogene 5: 1425-1430, 1990), which were subsequently isolated by cosmid rescue techniques, and further identified as a novel oncogene, termed cph because of its involvement in the carcinogenic progression of hamster embryo cells (Velasco et al, Oncogene 9: 2065-2069, 1994). We have analysed the expression of the cph proto-oncogene in adult Syrian hamster tissues by northern hybridization using cph-specific genomic probes. The three cph transcripts expressed in normal and neoplastic Syrian hamster embryo cells in culture (5.0, 3.5 and 2.0 kb) were also present in most adult tissues, although different mRNA species, most likely resulting from alternative splicing events, were expressed in testes. The highest steady-state level of cph mRNA was found in kidney, whereas cph expression was nearly undetectable in skin and skeletal muscle. Southern blot analyses of DNAs from other eucaryotic organisms were performed under moderate stringency conditions with a Syrian hamster-specific cph probe. Discrete cph-hybridizing sequences were present in genomes from yeast to mammalian species, including humans, thus demonstrating that cph is a highly conserved gene in eucaryotic evolution. Using fluorescence in situ hybridization (FISH), we have determined also the chromosomal localization of the cph proto-oncogene in the hamster genome. FISH experiments demonstrated that cph is a single copy gene, localized on the euchromatic short arm of the X chromosome, at region Xpa7. Because chromosome X is frequently involved in structural alterations in neoplastic Syrian hamster cells transformed by chemical carcinogens and oncogenic viruses, the localization of the cph locus on this chromosome supports the notion that the cph oncogene plays a role in the malignant conversion of chemically transformed hamster fibroblasts. The wide range of tissue-specific expression and species-specific distribution of cph strongly suggest that the normal function of the cph protein product(s) may be essential for metabolic processes involved in the regulation of cell proliferation and survival.

Animals↗
PubMed1996

Immortalization of human adult prostatic adenocarcinoma cells by human papilloma virus HPV16 and -18 DNA.

Primary prostate epithelial and prostate adenocarcinoma cells cultured in serum-free medium grew for up to 10 passages before senescence. Cells from prostate adenocarcinoma of a 55-year-old patient without lymph node involvement were transfected with plasmids containing recombinant human papilloma virus HPV16 or HPV18 DNA and the selectable neomycin-resistance gene. After G-418 selection, cells underwent crisis, and surviving cells infected with retroviruses encoding the HPV18 E6/E7 genes (HPV-PAC1), transfected with a head-to-tail dimer of the complete HPV16 genome (HPV-PAC2), or transfected with HPV18 E6/E7 early genes (HPV-PAC3) were established. HPV-PAC1 and HPV-PAC2 cultures appeared morphologically similar to primary cultures even after 40 passages. However, HPV-PAC2 cultures had a clonal morphology. All lines were positive for cytokeratin 18, had acquired vimentin expression, and contained either HPV16 or HPV18 sequences integrated into host DNA. None was tumorigenic in nude mice or formed colonies in soft agar. These cells did not secrete prostate specific antigen nor respond to androgen although tamoxifen inhibited the growth of the cells. Immunohistochemistry showed no evidence of p53 overexpression. Further characterization of these cell lines and examination of their response to chemotherapeutic agents may provide relevant information for the study of hormone-independent PC.

Adenocarcinoma↗
PubMed1995

Molecular cytogenetics of human papillomavirus-negative cervical carcinoma cell lines.

Cervical cancer is a malignancy closely associated with human papillomavirus (HPV). However, some cervical carcinomas occur in the absence of the virus. Two HPV-negative cervical carcinoma cell lines, C-33A and HT-3, were examined by G-banding and fluorescence in situ hybridization (FISH) using several whole chromosome and satellite probes. Combined conventional banding and FISH analysis significantly enhanced identification of complex and cryptic chromosome rearrangements. Common to both cell lines were translocations of chromosome 1, involving a specific site on the short arm and partial or complete loss of the short arm of chromosome 9, as well as loss of chromosome 13. The C-33A line had a relatively simple chromosomal constitution, with chromosome 1 being involved in translocations with chromosomes 9, 18, and 21. Karyotype reconstruction of G-banded and painted chromosomes indicate a net loss of DNA as a result of unbalanced translocations, which occurred only at site 9p24 and loss of one copy of chromosomes 13 and 14. In contrast, HT-3 cells had complex rearrangements and deletions of chromosomes 1p, 3p, 9p, 10q/p, 11p/q, and 17p, all regions with known tumor suppressor genes. The deletions observed in these HPV-negative cervical carcinomas will be important in delineating regions of tumor suppressor genes.

Carcinoma↗
PubMed1995

Transcriptional repression in normal human keratinocytes by wild-type and mutant p53.

Wild-type p53 is a nuclear phosphoprotein that inhibits cell proliferation and represses transcriptionally most TATA box-containing promoters in transformed or tumor-derived cell lines. This study demonstrates that p53 alters transcription of the long control region (LCR) of human papillomavirus type 18 (HPV-18). Wild-type and mutant p53 143Val to Ala repressed the HPV-18 LCR promoter in normal human keratinocytes, the natural host cell for HPV infections. Repression by wild-type p53 was also observed in C-33A cells and in an HPV-16-immortalized cell line with an inducible wild-type p53. However, when C-33A cells were cotransfected with the HPV-18 LCR and mutant 143Val to Ala, repression did not occur. Mutant p53 135Cys to Ser did not induce repression in either normal human keratinocytes or in the C-33A line; although like 143Val to Ala, it is thought to affect the DNA binding activity of the wild-type protein. The ability of mutant p53 143Val to Ala to inactivate the HPV early promoter in normal cells (by approximately 60% reduction) suggests that this mutant may be able to associate with wild-type p53 and interact with TATA box-binding proteins. Therefore, these results demonstrate that the transcriptional activities of p53 mutants may be dependent upon the cell type assayed and the form of its endogenous p53. Furthermore, normal human keratinocytes represent an alternative model for determining the activities of p53 mutants.

Cell Line↗
PubMed1995

Refined localization of the erbB-3 proto-oncogene by direct visualization of FISH signals on LUT-inverted and contrast-enhanced digital images of DAPI-banded chromosomes.

Contrast-enhanced, look-up-table (LUT)-inverted digital images of DAPI-banded chromosomes after fluorescence in situ hybridization (FISH) permit direct regional chromosomal localization of the fluorescent signals of single-copy gene probes. Improved quality and resolution of chromosome banding allowed a refined localization of the erbB-3 protooncogene from chromosome band 12q13 to sub-bands 12q13.2-13.3. This procedure can be used for direct and precise mapping of single-copy genes on both normal and cancer-cell-rearranged chromosomes.

Chromosome Banding↗
PubMed1995

Human herpesvirus 6 as a potential copathogen.

The molecular, biological and immunological studies of the recently identified human herpesvirus 6 (HHV-6) suggest that the virus is involved in the etiology of at least three lymphoproliferative diseases. Furthermore, HHV-6 may be an important cofactor in the pathogenesis of several other diseases, including HIV-associated disease and some cancers, but further investigation is needed to establish a causal relationship.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Integration site of human papillomavirus type-18 DNA in chromosome band 8q22.1 of C4-I cervical carcinoma: DNase I hypersensitivity and methylation of cellular flanking sequences.

The C4-I cell line derived from a non-keratinizing squamous cell carcinoma of the uterine cervix contains integrated human papillomavirus-18 DNA. Fluorescence in situ hybridization of C4-I cells demonstrated a single viral integration site at 8q22.1 on a derivative chromosome originating from an 8q;12q translocation. 8q22 is a site of chromosome fragility and is also recombinogenic in several human malignancies. DNase I hypersensitivity of the integration site was studied with a cellular flanking probe. A hypersensitive site was detected within 3 kb from viral DNA. The integration sites are undermethylated in C4-I and HeLa cells and fully methylated in tumor cell lines of other origin, such as lymphoid cells.

Carcinoma, Squamous Cell↗
PubMed1994

Chromosomal organization of viral integration sites in human papillomavirus-immortalized human keratinocyte cell lines.

The target specificity of viral integration is essential to determining the biologic significance of this integration to various pathologic conditions, including cancer. In this study the chromosomal features of several human papillomavirus (HPV)-16 integration sites mapped by in situ hybridization in human keratinocyte lines were visualized directly by G-banding and differential labeling with bromodeoxyuridine of later replicating domains. G-negative chromosomal bands exhibiting late replication were selectively targeted by HPV-16, suggesting that the structural and functional relationship of the state of chromatin condensation and replication is critical in accessibility to virus integration.

Attachment Sites, Microbiological↗
PubMed1994

Detection of human herpesvirus-6 in paraffin-embedded tissue of cervical cancer by polymerase chain reaction.

The polymerase chain reaction (PCR) was used to detect human herpesvirus-6 (HHV-6) DNA sequences from paraffin-embedded tissue from cervical cancer patients. Two of eight cases were positive for HHV-6 using two sets of HHV-6 primers. Hybridization of PCR products with specific radioisotope-labeled oligonucleotide probes confirmed the results. Furthermore, HHV-6 typing was possible by adapting restriction endonuclease digestion of PCR product. This method is useful for retrospective studies in investigating the etiologic role of HHV-6 in the development of human diseases.

Base Sequence↗
PubMed1994

Human herpesvirus 6 infects cervical epithelial cells and transactivates human papillomavirus gene expression.

To examine whether human herpesvirus 6 (HHV-6) is capable of infecting human cervical epithelial cells and altering expression of human papillomavirus (HPV) genes, HPV-immortalized or -transformed carcinoma cell lines were infected with HHV-6 variant A. No cytopathic effect was observed in infected cervical cells. However, immunofluorescence indicated that infected cells expressed early-late proteins of HHV-6 by day 3 postinfection. HHV-6 DNA was also detected by Southern blot hybridization after infection and persisted through continued subculture in an episomal state as proven by Gardella gel electrophoresis and fluorescence in situ hybridization. HHV-6 infection enhanced expression of HPV RNAs encoding the viral oncoproteins E6 and E7. Transient transfection assays showed that two HHV-6 molecular clones, pZVB-70 and pZVH-14, upregulated transcription 9- to 15-fold from a receptor plasmid containing the HPV type 18 regulatory sequences which control transcription in vivo. Cervical carcinoma cells infected with HHV-6 induced more rapid development of tumors in mice than did noninfected cells. These results are the first evidence that human cervical epithelial cells can be infected with HHV-6 and that HHV-6 contains transactivators which stimulate the HPV-transforming genes.

Animals↗
PubMed1994

Detection of human herpesvirus 6 and human papillomavirus 16 in cervical carcinoma.

A subset of human papillomaviruses (HPVs) is associated with the majority of cervical cancers; however, cofactors appear to be required for carcinogenic progression of HPV-induced neoplasia. As human herpesvirus-6 (HHV-6) was recently shown to infect cervical epithelial cells in vitro and activate transcription of HPV-transforming genes, human cervical dysplasia and cancers were analyzed for the presence of HHV-6 by multiple methods, including polymerase chain reaction, slot blot, Southern blot, and in situ hybridization. HHV-6 DNA sequences were detected in 6 of 72 cases of squamous cervical carcinoma and cervical intraepithelial neoplasia. HPV-16 was found in four of the HHV-6-positive cases (two squamous cervical carcinomas and two cervical intraepithelial neoplasias). None of the 30 normal cervices and biopsies of patients with cervicitis was positive for HHV-6 DNA. These results are the first suggestion of an in vivo association between HHV-6 and some cervical neoplasia.

Carcinoma, Squamous Cell↗
PubMed1994

cph, a novel oncogene which cooperates with H-ras in the transformation of NIH3T3 fibroblasts.

We have performed the molecular cloning of the non-ras transforming sequences previously detected in neoplastic Syrian hamster embryo fibroblasts initiated in vitro with 3-methylcholanthrene (MCA) (Notario et al., 1990). These sequences were isolated using cosmid-rescue techniques from a third-cycle NIH3T3 transformant obtained by sequential transfections of genomic DNA from MCA-initiated hamster fetal cells. Rescued (C-5) clones encompassed about 42.5 kbp of Syrian hamster genomic DNA containing hamster-specific repetitive elements (HRS). An internal 19 kbp BamHI fragment (B-1) was the only C-5 fragment which recognized specific transcripts in poly(A)+ RNA from hamster embryo cells. The same mRNA species were present in both normal and MCA-initiated neoplastic cells: a major transcript of about 2.5 kb, and other less abundant ones, ranging from approximately 2.0 kb to 5.0 kb. These mRNA species were detected consistently by each of several B-1 DNA subfragments located at positions spanning almost the entire B-1 length. The nucleotide sequence of some transcript-positive (S5P2 and S6) genomic B-1 fragments was determined. No significant homology exists between the nucleotide sequences of these B-1 subfragments and established DNA databases. Therefore, the C-5 cosmid clone contains novel genomic sequences. Transfection of C-5 DNA into mouse NIH3T3 cells resulted in the appearance of transformed foci (about five foci per microgram of DNA) within 25 days post-transfection, thus demonstrating the transforming activity of the C-5 clone, which was consequently renamed as the cph oncogene. Co-transfection of the cph oncogene with the human H-ras oncogene (T24), demonstrated a synergistic action between the two oncogenes in the transformation of murine fibroblasts.

3T3 Cells↗
PubMed1993

A Burkitt lymphoma cell line with integrated Epstein-Barr virus at a stable chromosome modification site.

Fluorescence in situ hybridization (FISH), Southern, and slot blotting were used to detect Epstein-Barr virus (EBV) DNA and RNA sequences in a Burkitt's lymphoma (BL) cell line derived from a North American patient (NAB-2). FISH analysis after hybridization with a BamHI "V" region of EBV showed that NAB-2 cells have EBV genome integrated at a single site on the short arm of chromosome 2(p13). Single hybridization signals were detected at homologous sites on both chromatoids and nuclei. Furthermore, hybridization of intact nuclei without formamide denaturation and heat allowed the detection of single specific viral RNA transcripts visible as "tracks" or "traces." Southern blot analysis confirmed the integration of EBV genome into the host DNA. Quantification of slot blot hybridization revealed that NAB-2 cells have on average one copy of EBV per cell. Virus insertion into chromosomal DNA caused a stable modification site expressed as a distinctive achromatic region adjacent to the band 2p13. The chromatid lesion at the site of EBV integration involving a recombinogenic and fragile site may have contributed to the development of the NAB-2 BL.

Blotting, Southern↗
PubMed1993

Cellular and molecular alterations in human epithelial cells transformed by recombinant human papillomavirus DNA.

Human papillomaviruses (HPVs) contribute to the development of benign and malignant cervical cancer; however, the exact role of papillomaviruses in the multistage carcinogenesis process is unclear. The development of HPV-immortalized cervical and foreskin cell lines represents a useful model for studying the role of HPVs in cervical cancer. Studies with these cells show that HPV genes regulate epithelial cell growth and differentiation. Transfection of HPV types associated with invasive cervical cancer results in immortalization of human epithelial cells, whereas HPVs not associated with cancer are ineffective. The combination of E6 and E7 genes, which are normally retained and expressed in cervical carcinomas, is sufficient for immortalization; however, the E7 gene alone induces immortality less efficiently. Although the immortalized cells actively express HPV oncoproteins observed in cervical cancer, after injection of immortal cells into nude mice, tumors are rare, having been reported only for HPV-18. Immortalized cells are resistant to terminal differentiation; in fact, HPVs may contribute to the carcinogenic process by uncoupling the processes of cell growth and differentiation. Host regulation of viral genes also is important in the malignant process. Endogenous cytokines modify HPV gene expression and influence the pathogenesis of HPV infection in the cervix. HPV gene expression is regulated by cellular transcriptional activators and repressors. This normal regulation is altered by viral integration. HPVs become integrated preferentially at chromosomal regions near fragile sites and protooncogenes. In fact, immortality is associated with induction of structural rearrangements frequently affecting HPV integration sites. Structural and numerical alterations nonrandomly involve chromosomes 1, 11, 19, and 20, with chromosome 1 alteration being the most predominant. Wild-type functions of Rb and p53 are necessary to control normal cell growth, and mutation or loss of these suppressor genes often contributes to cancer development. In HPV-containing carcinomas, pRb and p53 were wild type. However, in carcinomas lacking HPV, both suppressor genes were mutated. Functional inactivation of these tumor suppressor genes by HPV oncoproteins E6 and E7 may explain this difference. Treatment of HPV-immortalized cells with ras or a subfragment of herpes simplex virus (HSV) of HPV-immortalized cells resulted in locally invasive carcinomas when the cells were implanted subcutaneously in nude mice. These experiments indicate that HPV integration and expression are insufficient for malignancy but that HPVs do participate in the multistep development of cancer.

Animals↗
PubMed1992

Down-regulation of keratin 14 gene expression after v-Ha-ras transfection of human papillomavirus-immortalized human cervical epithelial cells.

Keratin expression in human cervical squamous cell carcinoma (SCC) lines differed significantly from both normal and human papillomavirus (HPV) immortalized exocervical cells. Keratin 14 (K14) expression, determined by protein synthesis and mRNA levels, was dramatically down-regulated in the cervical SCC lines while keratin 5 (K5) expression was not. K14 expression was similarly down-regulated in an HPV-16 immortalized cervical cell line after tumorigenic transformation with recombinant v-Ha-ras DNA. Cultures derived from nude mouse tumor explants also exhibited an altered keratin profile and the levels of K14 protein synthesis, as well as K14 mRNA, were not detectable. In both cases K5 protein synthesis was not significantly down-regulated. In addition, neoplastic cervical SCC lines exhibited up-regulation of keratins 7, 8, 13, and 19, combined with slight down-regulation of keratins 6 and 16. Epidermal keratinocytes responded in a different manner to exocervical cells. Transfection of human papillomavirus-immortalized epidermal keratinocytes with the BglII N fragment of herpes simplex virus 2 produced a neoplastic cell line, but K5 and K14 expression remained unchanged. Thus, neoplastic transformation of human exocervical cells, both in vivo (spontaneous cervical SCC) and in vitro (HPV-16- and v-Ha-ras-induced cervical SCC), is accompanied by characteristic changes in keratin expression. The specific down-regulation of K14 in these tumorigenic cervical cells, in the absence of significant changes in the expression of K5, implies that the normal coordinate regulation of K5 and K14 gene expression has been uncoupled.

Blotting, Northern↗
PubMed1992

Leukoregulin and gamma-interferon inhibit human papillomavirus type 16 gene transcription in human papillomavirus-immortalized human cervical cells.

The human papillomavirus (HPV) transforming genes E6 and E7 are retained and expressed in the majority of cervical cancers implying an important role for these proteins in maintenance of the malignant phenotype. Leukoregulin (LR) and recombinant gamma-interferon (r-IFN-gamma), lymphokines secreted by immune cells present in regressing HPV infections, inhibited transcription of E6/E7 RNAs in several human cervical epithelial cell lines immortalized by recombinant HPV-16, -18, and -33 DNAs. r-IFN alpha was not effective. Reduction in E6/E7 RNA expression was accompanied by inhibition of cell proliferation coincident with an increase in epidermal transglutaminase activity, a marker of squamous differentiation. LR and r-IFN gamma enhanced transcription of class 1 cell surface histocompatibility antigens (HLA) and r-IFN gamma additionally induced HLA class 2 expression. HPV-immortalized cells developed partial resistance to the growth inhibitory effects of lymphokines after malignant transformation or extended propagation in culture. This is the first demonstration that LR and r-IFN gamma selectively inhibit transcription of HPV-transforming genes and suggests a molecular mechanism by which these lymphokines participate in regression of premalignant cells.

Blotting, Northern↗